Differentiating salmonid migratory ecotypes through stable isotope analysis of collagen: Archaeological and ecological applications

The ability to distinguish between different migratory behaviours (e.g., anadromy and potamodromy) in fish can provide important insights into the ecology, evolution, and conservation of many aquatic species. We present a simple stable carbon isotope (δ13C) approach for distinguishing between sockeye (anadromous ocean migrants) and kokanee (potamodromous freshwater residents), two migratory ecotypes of Oncorhynchus nerka (Salmonidae) that is applicable throughout most of their range across coastal regions of the North Pacific Ocean. Analyses of kokanee (n = 239) and sockeye (n = 417) from 87 sites spanning the North Pacific (Russia to California) show that anadromous and potamodromous ecotypes are broadly distinguishable on the basis of the δ13C values of their scale and bone collagen. We present three case studies demonstrating how this approach can address questions in archaeology, archival, and conservation research. Relative to conventional methods for determining migratory status, which typically apply chemical analyses to otoliths or involve genetic analyses of tissues, the δ13C approach outlined here has the benefit of being non-lethal (when applied to scales), cost-effective, widely available commercially, and should be much more broadly accessible for addressing archaeological questions since the recovery of otoliths at archaeological sites is rare.

. Primers used in this study.
PCR amplifications were performed in a Mastercycler Gradient (Eppendorf, Mississauga, ON) thermocycler in a 30 μL reaction containing 1.5× PCR Gold Buffer (Applied Biosystems, Carlsbad, CA, USA), 2 mM MgCl2, 0.2 mM of each dNTP, 0.45 μM of primers sdY-F19 and sdY-R20 (clock1a/sdY assay) or 0.6 μM of primers sdY-F19 and sdY-R20 (D-loop/sdY assay), 0.3 μM of primers Clk1a-F50 and Clk1a-R60 (clock1a/sdY assay) or 0.6 μM of primers Smc7 and Smc8 (D-loop/sdY assay), 1 mg/mL BSA, 3 μL DNA solution, and 1 U AmpliTaq Gold (Applied Biosystems, Carlsbad, CA). The thermocycling program for both PCR sex identification assays consisted of an initial denaturation step at 95 °C for 12 min followed by 60 cycles at 95 °C for 30 s (denaturation), 54 °C for 30 s (annealing), and 70 °C for 40 s (extension), and a final extension step at 72 °C for 7 min. To detect instances of contamination, a negative PCR control was included in each PCR run and each assay was applied to the blank extraction controls. All PCR and post-PCR procedures were conducted in a laboratory physically separated from the aDNA laboratory.
Following amplification, 5 μl of PCR product from each sample was pre-stained with SYBR Green I (Life Technologies, Carlsbad, CA), electrophoresed on a 3% agarose gel, and visualized with a Dark Reader transilluminator (Clare Chemical Research, Dolores, CO). Sex identities were assigned to the samples through a visual analysis of the electrophoresis gels using the criteria established by Royle and colleagues (2018). In brief, a sample was identified as male if sdY was successfully amplified with both assays, while a female identity was assigned to a sample if sdY was not amplified with either assay but both IPCs were amplified. No sex identity was assigned to a sample if the assays yielded discordant results or if one of the assays failed to amplify DNA.

Species Identification
Following Royle and colleagues (2018), we sought to assign species identifications to the samples by sequencing the D-loop fragment co-amplified as an IPC in the D-loop/sdY sex identification assay. To confirm the species identifications assigned to the samples, we also sequenced a 168 bp fragment of cytochrome b, which was amplified in a singleplex PCR with primers CytB5 and CytB6 (Table 1) (Royle, et al. 2020;Yang, et al. 2004). To improve sequencing quality, D-loop was also amplified from a single sample (IUBC 5226) through a singleplex PCR with the same primers (Smc7 and Smc8) used in the D-loop/sdY sex identification assay.
Singleplex PCR amplifications were performed in a Mastercycler Gradient or Personal thermocycler (Eppendorf, Mississauga, ON) in a 30 μL reaction volume that included 1.5× PCR Gold Buffer (Applied Biosystems, Carlsbad, CA), 2 mM MgCl2, 0.2 mM of each dNTP, 0.3 μM of each cytochrome b or D-loop primer, 1 mg/mL BSA, 1.5-3 μL DNA solution, and 0.75-1 U AmpliTaq Gold (Applied Biosystems, Carlsbad, CA). The thermocycling program for the singleplex PCRs consisted of an initial denaturation step at 95 °C for 12 min followed by 60 cycles at 95 °C for 30 s (denaturation), 54 °C for 30 s (annealing), and 70 °C for 40 s (extension), and a final extension step at 72 °C for 7 min. Following amplification, 5 μl of PCR product from each sample was pre-stained with SYBR Green I (Life Technologies, Carlsbad, CA), electrophoresed on a 2-3% agarose gel, and visualized with a Dark Reader transilluminator (Clare Chemical Research, Dolores, CO). A negative PCR control was included in each singleplex PCR run in order to monitor for contamination. Singleplex PCRs were also performed on both blank extraction controls.
Successfully amplified D-loop and cytochrome b fragments were directly sequenced with the forward or reverse amplification primers at Eurofins Genomics (Toronto, ON). Prior to sequencing, the PCR products obtained from some of the samples were purified with ExoSAP-IT Express (Life Technologies, Carlsbad, CA) following the manufacturer's instructions. The obtained sequences were visually edited, truncated to remove the primer sequences, and compiled in ChromasPro v 2.1.8 (http://technelysium.com.au). To determine their closest taxonomic match, the edited sequences were compared against reference sequences accessioned in GenBank (Sayers, et al. 2019) through a BLASTn search (Altschul, et al. 1990). Multiple alignments of the ancient cytochrome b and D-loop sequences and reference sequences from 8 Oncorhynchus species (O. clarkii, O. gorbuscha, O. keta, O. kisutch, O. masou, O. mykiss, O. nerka, and O. tshawytscha) as well as Salmo salar were performed with Clustal W (Thompson, et al. 1994) through BioEdit v 7.2.5 (Hall, 1999). The resulting alignment was visually examined in BioEdit and the sequences were trimmed to the same length. For each marker, neighbourjoining trees were constructed in MEGA X (Kumar, et al. 2018) using a Kimura 2-parameter substitution model and 1000 bootstrap replications. Species-level identifications were assigned to samples if the obtained cytochrome b and D-loop sequences matched or closely resembled sequences from a single species and differed from closely related species (Yang, et al. 2004) The tree was rooted using an Atlantic salmon (Salmo salar) sequence as an outgroup. The numbers at nodes denote the bootstrap values for nodes with ≥50% support after 1000 replications. The scale bar represents the number of nucleotide substitutions per site.

Fig 2. Neighbour-joining tree displaying the phylogenetic relationship between the D-loop sequences obtained from the archaeological salmonid samples analyzed in this study (denoted with filled squares; aDNA [ELS#] and isotope [IUBC #] lab numbers provided) and
Oncorhynchus reference sequences (GenBank accession numbers shown). The tree was rooted using an Atlantic salmon (Salmo salar) sequence as an outgroup. The numbers at nodes denote the bootstrap values for nodes with ≥50% support after 1000 replications. The scale bar represents the number of nucleotide substitutions per site